Purification and characterization of a novel proline-directed protein kinase from bovine brain.

摘要: A novel protein kinase which phosphorylates a synthetic peptide substrate (RRPDAHRTPNRAF) has been purified approximately 200,000-fold from bovine brain. This contains the consensus sequence for phosphorylation by p34cdc2 kinase. The purification procedure took advantage of phenomenon that this brain kinase, in partially extracts, chromatographed on gel filtration column as high molecular weight complex dissociated buffer containing 1 M NaCl. native enzyme was estimated to be 63,000, and displayed two bands M(r) = 33,000 25,000 sodium dodecyl sulfate-polyacrylamide electrophoresis. On Western immunoblot, reacted strongly with antibodies specific conserved amino-terminal sequence, weakly PSTAIRE not at all carboxyl terminus, HeLa cell p34cdc2. were similar displaying good activity toward parent substrate, but no analogues -T-P- motif substituted either -T-G- or -T-A-. Both kinases showed marked preference phosphorylating derived H1 histone (KTPKKAKKPKTPKKAKKL), both could phosphorylated src-family tyrosine p56lyn, spleen. However, did co-purify subunit having corresponding known cyclins, nor it undergo interaction p13suc1 beads, suggesting is distinct